Conditioned medium from cells cultured under hypoxic conditions and uses thereof

ABSTRACT

The production and use of extracellular matrix or conditioned medium compositions and more specifically to compositions obtained by culturing cells under hypoxic conditions on a surface in a suitable growth medium.

FIELD

The present invention relates to the production and use of extracellularmatrix or conditioned medium compositions and more specifically tocompositions obtained by culturing cells under hypoxic conditions on asurface in a suitable growth medium. Use of the composition is alsocontemplated.

BACKGROUND OF THE INVENTION

Conditioned medium from in vitro studies and their use in a variety oftherapeutic and medical applications have been described in the art. Onetherapeutic application of extracellular matrix (ECM) compositionsincludes the treatment and repair of soft tissue and skin defects suchas wrinkles and scars.

The conditioned medium compositions may include amino acids, salts,vitamins, minerals sugars, lipids, nucleosides and proteins. Theconditioned medium may contain many of the original components of thecell culture medium as well as a variety of cellular metabolites andsecreted proteins including, for example, biologically active growthfactors, inflammatory mediators and other extracellular proteins.

The repair or augmentation of soft tissue defects caused by acne,surgical scarring or aging has proven to be very difficult. A number ofmaterials have been used to correct soft tissue defects with varyingdegrees of success, however, no material has been completely safe andeffective. For example, silicon causes a variety of physiological andclinical problems including long term side effects such as nodules,recurring cellulitis and skin ulcers.

Accordingly, new materials are needed for soft tissue repair andtreatment of soft tissue that may overcome the deficiencies of priormaterials.

In vitro cultured medium can also be used to treat damaged soft tissuesuch as dermal tissue, for example.

In vitro cultured medium can additionally be used to repair and/orregenerate damaged cells or tissue such as dermal tissue, for example.The compositions of the present invention may be useful for treatment oftrauma to the skin such as burns, laser treatment, and the effects ofaging. Effects of aging may include skin discoloration, uneven skintexture, roughness and uneven skin tone, for example.

Fueled in part by the stem cell revolution, tissue engineeringtechnology offers the promise of tissue regeneration and replacementfollowing trauma or treatment of degenerative diseases. It can also beused in the context of cosmetic procedures.

Tissue engineering techniques can be used to generate both autologousand heterologous tissue or cells using a variety of cell types andculture techniques. In creating an autologous implant, donor tissue maybe harvested and dissociated into individual cells, and subsequentlyattached and cultured on a substrate to be implanted at the desired siteof the functioning tissue. Many isolated cell types can be expanded invitro using cell culture techniques, however, anchorage dependent cellsrequire specific environments, often including the presence of athree-dimensional scaffold, to act as a template for growth.

Thus, the development of natural materials that are suitable for topicalapplication is greatly needed.

SUMMARY

The embodiments herein encompass methods and compositions comprisinghuman fibroblast-derived cell culture medium that is cultured underhypoxic conditions.

In a first aspect, a method of producing a composition for improvementof tissue in a subject is provided, the method comprising: culturinghuman fibroblast cells under hypoxic conditions on microcarrier beads ora three dimensional surface in a suitable cell culture medium under 1-5%oxygen, thereby simulating the early embryonic environment, andgenerating a composition with embryonic-like proteins that promotes therepair and regeneration of damaged tissue when administered to theregion of tissue in need of repair. In some embodiments, the methodfurther comprises adding at least one botanical to the composition. Insome embodiments, the method further comprises adding at least oneextract to the composition. In some embodiments, the method furthercomprises adding at least one peptide to the composition. In someembodiments, the culturing is performed on microcarrier beads or athree-dimensional surface in a suitable cell culture medium. In someembodiments, the culturing is performed for at least two weeks. In someembodiments, the conditioned medium is collected after two weeks. Insome embodiments, the method further comprises adding a seed extract tothe composition. In some embodiments, the method further comprisesadding a marine extract to the composition. In some embodiments, themethod further comprises adding a bacterial ferment to the composition.In some embodiments, the conditioned medium has embryonic-like proteins.In some embodiments, the conditioned medium further comprises cytokines.In some embodiments, the conditioned medium further comprises at leastone matrix protein. In some embodiments, the at least one peptidecomprises dimer tripeptide 43 and/or trifluoroacetyl tripeptide-2. Insome embodiments, the subject is in need of tissue repair. In someembodiments, the subject has fine and or deep wrinkles. In someembodiments, the subject exhibits tactile and/or skin roughness of thetissue. In some embodiments, the subject has hyperpigmentation. In someembodiments, the subject has photodamage. In some embodiments, thesubject lacks evenness in pigmentation and/or skin tone. In someembodiments, the subject has a skin coloring on the Fitzpatrick scale of1, 2, 3, 4 or 5.

In a second aspect, a composition made by the method of any one of theembodiments described herein is provided for use in treating a subject.

In a third aspect, a composition is prepared from a conditioned mediumthat is prepared from a cell culture medium cultured with embryonic-likeproteins under hypoxic conditions, at least one botanical, at least oneextract and at least one peptide. In some embodiments, the compositionis odorless. In some embodiments, the composition is clear.

In a fourth aspect, a method of improving the appearance of anindividual is provided, the method comprising: topically applying acomposition of any one of the embodiments described herein onto thesurface of a subject's skin to thereby improve the aesthetic quality ofthe skin. In some embodiments, the subject has fine or deep wrinkles. Insome embodiments, the subject has tactile and/or skin roughness of thetissue. In some embodiments, the subject has loose or sagging skin. Insome embodiments, the method improves roughness of skin. In someembodiments, the method restores volume moisture to the tissue. In someembodiments, the method results in reduced inflammatory response of thetissue. In some embodiments, the subject is in need of tissue repair. Insome embodiments, the subject is suffering from a burn wound. In someembodiments, the method leads to healing of the tissue. In someembodiments, the skin is protected from free radical damage. In someembodiments, the composition supports epidermal cell-cell adhesion. Insome embodiments, the composition supports a dermal epidermal junction.In some embodiments, the composition supports stem cells function andproliferation. In some embodiments, the composition supportsintercellular communication. In some embodiments, the compositionsupports cellular recycling and protein homeostasis. In someembodiments, the composition prevents cellular senescence. In someembodiments, the composition supports collagen, elastin and other ECMcomponents. In some embodiments, the composition supports heparansulfate and proteoglycans.

DETAILED DESCRIPTION

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Where the definition of terms as used in the specification departs fromthe commonly used meaning of the term, applicant intends to utilize thedefinitions provided herein, unless specifically indicated.

Treating soft tissue defects that may arise from aging has proven to bequite challenging. A number of materials have been used to correct softtissue defects with varying degrees of success, however, no material hasbeen found to be completely safe and effective. For example, siliconcauses a variety of physiological and clinical problems including longterm side effects, such as nodules, recurring cellulitis and skinulcers.

Aging leads to defects including but not limited to fine lines and deepwrinkles, hyperpigmentation, elastin degradation, collagen degradation,inflammation, and decreased fibroblast function.

In some embodiments, a method for making compositions that include oneor more proteins that are expressed during hypoxic conditions isprovided. These proteins may be embryonic proteins, for example. Inparticular, the compositions are generated by culturing cells underhypoxic conditions on a surface (e.g., two-dimensional orthree-dimensional) in a suitable growth medium. The culturing methodproduces both soluble and non-soluble fractions which may be usedseparately or in combination to obtain physiologically acceptablecompositions having a variety of applications. The hypoxic conditionsmay induce proteins that stimulate precursor cells and may furtherstrengthen the skin rejuvenating benefits of human fibroblast-derivedgrowth factors.

In certain embodiments, a composition for repair and regeneration ofskin tissue on a subject is disclosed comprising a conditioned mediumcollected from culturing human fibroblast cells under hypoxic conditionsin a suitable cell culture medium and at least one additive, wherein theconditioned medium is stored in a first container and the additive isstored in a second container, and wherein the conditioned medium and theadditive are mixed prior to application to the skin tissue. The firstcontainer and the second container are separate chambers in a dualchamber container.

In certain embodiments, the human fibroblast cells are cultured underhypoxic conditions on a substrate grown in a suitable cell culturemedium under 1-5% oxygen thereby producing embryo-like properties. Thesubstrate may be microcarrier beads or a three-dimensional surface.

The compositions of the present invention may be colorless and furthercomprise at least one botanical or botanical extract in a range of about0.5 to about 2.0% by weight of the composition, at least one peptidepresent within a range of about 0.0001 to about 0.001% by weight of thecomposition, at least one seed extract present within a range of about0.5 to about 2% by weight of the composition, at least one marineextract within a range of about 0.01 to about 0.1% by weight of thecomposition, at least one bacterial ferment present within a range ofabout 0.5 to about 3.0% by weight of the composition, stem cell factorspresent within a range of about 0.1 to about 30% by weight of thecomposition, and/or cytokines.

In some embodiments, the composition may include cellular cytokines andgrowth factors. Extracellular proteins may be secreted into conditionedcell media such as growth factors, cytokines, and stress proteins. Thesemay be used in the preparation of products for use in a large variety ofareas including tissue repair, e.g., in the treatment of wounds andother tissue defects such as cosmetic defects as well as human andanimal feed supplements. For example, growth factors are known to playan important role in the wound healing process. In general, it isthought desirable in the treatment of wounds to enhance the supply ofgrowth factors by direct addition of these factors.

Cellular cytokines and growth factors are involved in a number ofcritical cellular processes including cell proliferation, adhesion,morphologic appearance, differentiation, migration, inflammatoryresponses, angiogenesis, and cell death. Studies have demonstrated thathypoxic stress and injury to cells induce responses including increasedlevels of mRNA and proteins corresponding to growth factors such as PDGF(platelet-derived growth factor), VEGF (vascular endothelial growthfactor), FGF (fibroblast growth factor), and IGF (insulin-like growthfactor) (Gonzalez-Rubio, M. et al., 1996, Kidney Int 50(1):164-73;Abramovitch, R. et al., 1997, Int J. Exp. Pathol. 78(2):57-70; Stein, I.et al., 1995, Mol Cell Biol. 15(10):5363-8; Yang, W. et al., 1997, FEBSLett. 403(2):139-42; West, N. R. et al., 1995, J. Neurosci. Res.40(5):647-59).

The growth factors are naturally secreted proteins which may regulate avariety of cell functions.

In some embodiments, the composition includes proteins that stimulateprecursor cells.

The compositions of the present invention have a variety of applicationsincluding, but not limited to, promoting repair and/or regeneration ofdamaged cells or tissues, use in patches and implants to promote tissueregeneration (e.g., hernial repair, pelvic floor repair, rotator cuffrepair, and wound repair), use in tissue culture systems for culturingcells, such as stem cells, use in surface coatings used in associationwith implantable devices (e.g., pacemakers, stents, stent grafts,vascular prostheses, heart valves, shunts, drug delivery ports orcatheters, hernial and pelvic floor repair patches), promoting softtissue repair, augmentation, and/or improvement of a skin surface, suchas wrinkles, use as a biological anti-adhesion agent or as a biologicalvehicle for cell delivery or maintenance at a site of delivery.

In some embodiments, compositions comprising the cultured medium may beused for the treatment of wrinkles (deep wrinkles, fine lines), woundhealing, scar healing, hyperpigmentation, uneven skin tone, dry skin,tactile and/or skin roughness, and/or treatment of scarring.

The embodiments described herein are based in part, on the discoverythat cells cultured on three-dimensional surfaces under conditions thatstimulate the early embryonic environment (hypoxia and reducedgravitational forces) prior to angiogenesis produces ECM compositionswith fetal properties, including generation of embryonic proteins.Growth of cells under hypoxic conditions demonstrate a unique ECM withfetal properties and growth factor expression. Unlike the culturing ofECM under traditional culture conditions, over 5000 genes aredifferentially expressed in ECM cultured under hypoxic conditions. Thisresults in a cultured ECM that has different properties and a differentbiological composition. For example, an ECM produced under hypoxicconditions is similar to fetal mesenchymal tissue in that it isrelatively rich in collagens type III, IV, and V, and glycoproteins suchas fibronectin, SPARC, thrombospondin, and hyaluronic acid.

In some embodiments, the hypoxic conditions induce proteins thatstimulate precursor cells.

Hypoxia also enhances expression of factors which regulate wound healingand organogenesis, such as VEGF, FGF-7, and TGF-β, as well as multipleWnt factors including writs 2b, 4, 7a, 10a, and 11. Cultured embryonichuman ECM also stimulates an increase of metabolic activity in humanfibroblasts in vitro, as measured by increased enzymatic activity.Additionally, there is an increase in cell number in response to thecultured embryonic ECM.

Before the present compositions and methods are described, it is to beunderstood that embodiments described herein are not limited toparticular compositions, methods, and experimental conditions described,as such compositions, methods, and conditions may vary. It is also to beunderstood that the terminology used herein is for purposes ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyin the appended claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to “themethod” includes one or more methods, and/or steps of the type describedherein which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

In some embodiments, methods for making compositions comprising one ormore embryonic-like proteins and applications thereof are provided. Inparticular, the compositions are generated by culturing cells underhypoxic conditions on a two-dimensional or three-dimensional surface ina suitable cell culture medium. The compositions are derived by growingcells on a three-dimensional framework resulting in a multi-layer cellculture system. Cells grown on a three-dimensional framework support, inaccordance with the present invention, grow in multiple layers, forminga cellular matrix. Growth of the cultured cells under hypoxic conditionsresults in differential gene expression as the result of hypoxicculturing conditions versus traditional culture.

An ECM is a composition of proteins and biopolymers that substantiallycomprise tissue that is produced by cultivation of cells. Stromal cells,such as fibroblasts, are an anchorage dependent cell type requiringgrowth while attached to materials and surfaces suitable for cellculture. The ECM produced by the cultured cells are deposited in athree-dimensional arrangement providing spaces for the formation oftissue-like structures.

The cultivation materials providing three-dimensional architectures arereferred to as scaffolds. Spaces for deposition of the ECM are in theform of openings within, for example woven mesh or interstitial spacescreated in a compacted configuration of spherical beads, calledmicrocarriers.

As used herein, “ECM” or “extracellular matrix composition” includessoluble and non-soluble fractions or any portion thereof The non-solublefraction of an ECM includes those secreted proteins and biologicalcomponents that are deposited on the support or scaffold. The solublefraction includes refers to culture media in which cells have beencultured and into which the cells have secreted active agent(s) andincludes those proteins and biological components not deposited on thescaffold. Both fractions may be collected, and optionally furtherprocessed, and used individually or in combination in a variety ofapplications as described herein.

The three-dimensional support or scaffold used to culture stromal cellsmay be of any material and/or shape that: (a) allows cells to attach toit (or can be modified to allow cells to attach to it); and (b) allowscells to grow in more than one layer (i.e., form a three dimensionaltissue). In some embodiments, a substantially two-dimensional sheet ormembrane or beads may be used to culture cells that are sufficientlythree dimensional in form.

The biocompatible material is formed into a three-dimensional structureor scaffold, where the structure has interstitial spaces for attachmentand growth of cells into a three-dimensional tissue. The openings and/orinterstitial spaces of the framework in some embodiments are of anappropriate size to allow the cells to stretch across the openings orspaces. Maintaining actively growing cells stretched across theframework appears to enhance production of the repertoire of growthfactors responsible for the activities described herein. If the openingsare too small, the cells may rapidly achieve confluence but be unable toeasily exit from the mesh. These trapped cells may exhibit contactinhibition and cease production of the appropriate factors necessary tosupport proliferation and maintain long term cultures. If the openingsare too large, the cells may be unable to stretch across the opening,which may lead to a decrease in stromal cell production of theappropriate factors necessary to support proliferation and maintain longterm cultures. Typically, the interstitial spaces are at least about 100um, at least about 140 um, at least about 150 um, at least about 180 um,at least about 200 um, or at least about 220 um. When using a mesh typeof matrix, as exemplified herein, the openings range from about 100 μmto about 220 μm. However, depending upon the three-dimensional structureand intricacy of the framework, other sizes are permissible. Any shapeor structure that allows the cells to stretch and continue to replicateand grow for lengthy time periods may function to elaborate the cellularfactors in accordance with the methods herein.

In some aspects, the three dimensional framework is formed from polymersor threads that are braided, woven, knitted or otherwise arranged toform a framework, such as a mesh or fabric. The materials may also beformed by casting of the material or fabrication into a foam, matrix, orsponge-like scaffold. In other aspects, the three-dimensional frameworkis in the form of matted fibers made by pressing polymers or otherfibers together to generate a material with interstitial spaces. Thethree-dimensional framework may take any form or geometry for the growthof cells in culture. Thus, other forms of the framework, as furtherdescribed below, may suffice for generating the appropriate conditionedmedium.

A number of different materials may be used to form the scaffold orframework. These materials include non-polymeric and polymericmaterials. Polymers, when used, may be any type of polymer, such ashomopolymers, random polymers, copolymers, block polymers, coblockpolymers (e.g., di, tri, etc.), linear or branched polymers, andcrosslinked or non-crosslinked polymers. Non-limiting examples ofmaterials for use as scaffolds or frameworks include, among others,glass fibers, polyethylenes, polypropylenes, polyamides (e.g., nylon),polyesters (e.g., dacron), polystyrenes, polyacrylates, polyvinylcompounds (e.g., polyvinylchloride; PVC), poly carbonates,polytetrafluorethylenes (PTFE; TEFLON), thermanox (TPX), nitrocellulose,polysaacharides (e.g., celluloses, chitosan, agarose), polypeptides(e.g., silk, gelatin, collagen), polyglycolic acid (PGA), and dextran.

In some embodiments, the framework or beads may be made of materialsthat degrade over time under the conditions of use. Biodegradable alsorefers to absorbability or degradation of a compound or composition whenadministered in vivo or under in vitro conditions. Biodegradation mayoccur through the action of biological agents, either directly orindirectly. Non-limiting examples of biodegradable materials include,among others, polylactide, polyglycolide, poly(trimethylene carbonate),poly(lactide-co-glycolide) (i.e., PLGA), polyethylene terephtalate(PET), polycaprolactone, catgut suture material, collagen (e.g., equinecollagen foam), polylactic acid, or hyaluronic acid. For example, thesematerials may be woven into a three-dimensional framework such as acollagen sponge or collagen gel.

In other aspects, where the cultures are to be maintained for longperiods of time, cryopreserved, and/or where additional structuralintegrity is desired, the three dimensional framework may be comprisedof a nonbiodegradable material. As used herein, a nonbiodegradablematerial refers to a material that does not degrade or decomposesignificantly under the conditions in the culture medium. Exemplarynondegradable materials include, as non-limiting examples, nylon,dacron, polystyrene, polyacrylates, polyvinyls, polytetrafluoroethylenes(PTFE), expanded PTFE (ePTFE), and cellulose. An exemplary nondegradingthree dimensional framework comprises a nylon mesh, available under thetradename Nitex®, a nylon filtration mesh having an average pore size of140 μm and an average nylon fiber diameter of 90 μm (#3-210/36, Tetko,Inc., N.Y.).

In other aspects, the beads, scaffold or framework is a combination ofbiodegradeable and non-biodegradeable materials. The non-biodegradablematerial provides stability to the three dimensional scaffold duringculturing while the biodegradeable material allows formation ofinterstitial spaces sufficient for generating cell networks that producethe cellular factors sufficient for therapeutic applications. Thebiodegradable material may be coated onto the non-biodegradable materialor woven, braided or formed into a mesh. Various combinations ofbiodegradable and non-biodegradable materials may be used. An exemplarycombination is poly(ethylene therephtalate) (PET) fabrics coated with athin biodegradable polymer film, poly[D-L-lactic-co-glycolic acid), inorder to obtain a polar structure.

In various aspects, the scaffold or framework material may bepre-treated prior to inoculation with cells to enhance cell attachment.For example, prior to inoculation with cells, nylon screens in someembodiments are treated with 0.1 M acetic acid, and incubated inpolylysine, fetal bovine serum, and/or collagen to coat the nylon.Polystyrene could be similarly treated using sulfuric acid. In someembodiments, the growth of cells in the presence of thethree-dimensional support framework may be further enhanced by adding tothe framework or coating it with proteins (e.g., collagens, elastinfibers, reticular fibers), glycoproteins, glycosaminoglycans (e.g.,heparan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatansulfate, keratan sulfate, etc.), fibronectins, and/or glycopolymer(poly[N-p-vinylbenzyl-D-lactoamide], PVLA) in order to improve cellattachment. Treatment of the scaffold or framework is useful where thematerial is a poor substrate for the attachment of cells.

In one aspect, mesh is used for production of ECM. The mesh is a wovennylon 6 material in a plain weave form with approximately 100 μmopenings and approximately 125 μm thick. In culture, fibroblast cellsattach to the nylon through charged protein interactions and grow intothe voids of the mesh while producing and depositing ECM proteins. Meshopenings that are excessively large or small may not be effective butcould differ from those above without substantially altering the abilityto produce or deposit ECM. In another aspect, other woven materials areused for ECM production, such as polyolefin's, in weave configurationsgiving adequate geometry for cell growth and ECM deposition.

For example, nylon mesh is prepared for cultivation in any of the stepsof the invention by cutting to the desired size, washing with 0.1-0.5Macetic acid followed by rinsing with high purity water and then steamsterilized. For use as a three-dimensional scaffold for ECM productionthe mesh is sized into squares approximately 10 cm×10 cm. However, themesh could be any size appropriate to the intended application and maybe used in any of the methods of the present invention, includingcultivation methods for inoculation, cell growth and ECM production andpreparation of the final form.

In other aspects, the scaffold for generating the cultured tissues iscomposed of microcarriers, which are beads or particles. The beads maybe microscopic or macroscopic and may further be dimensioned so as topermit penetration into tissues or compacted to form a particulargeometry. In some embodiments of any one of each or any of the above- orbelow-mentioned embodiments of the tissue penetrating embodiments, theframework for the cell cultures comprises particles that, in combinationwith the cells, form a three dimensional tissue. The cells attach to theparticles and to each other to form a three dimensional tissue. Thecomplex of the particles and cells is of sufficient size to beadministered into tissues or organs, such as by injection or catheter.Beads or microcarriers are typically considered a two-dimensional systemor scaffold.

As used herein, a “microcarrier” refers to a particle having a size inthe range of of nanometers to micrometers in any shape or geometry,including but not limited to being irregular, non-spherical, spherical,or ellipsoid.

The size of the microcarriers suitable for the purposes herein can be ofany size suitable for the particular application. In some embodiments,the size of microcarriers suitable for the three dimensional tissues maybe those administrable by injection. In some embodiments, themicrocarriers have a particle size range of at least about 1μm, at leastabout 10 μm, at least about 25 μm, at least about 50 μm, at least about100 μm, at least about 200 μm, at least about 300 μm, at least about 400μm, at least about 500 μm, at least about 600 μm, at least about 700 μm,at least about 800 μm, at least about 900 μm, at least about 1000 μm.

In some embodiments, the microcarriers are made of biodegradablematerials. In some embodiments, microcarriers comprising two or morelayers of different biodegradable polymers may be used. In someembodiments, at least an outer first layer has biodegradable propertiesfor forming the three dimensional tissues in culture, while at least abiodegradable inner second layer, with properties different from thefirst layer, is made to erode when administered into a tissue or organ.

In some embodiments, the microcarriers are porous microcarriers. Porousmicrocarriers refer to microcarriers having interstices through whichmolecules may diffuse in or out from the microparticle. In someembodiments, the microcarriers are non-porous microcarriers. A nonporousmicroparticle refers to a microparticle in which molecules of a selectsize do not diffuse in or out of the microparticle.

Microcarriers for use in the compositions are biocompatible and have lowor no toxicity to cells. Suitable microcarriers may be chosen dependingon the tissue to be treated, type of damage to be treated, the length oftreatment desired, longevity of the cell culture in vivo, and timerequired to form the three dimensional tissues. The microcarriers maycomprise various polymers, natural or synthetic, charged (i.e., anionicor cationic) or uncharged, biodegradable, or nonbiodegradable. Thepolymers may be homopolymers, random copolymers, block copolymers, graftcopolymers, and branched polymers.

In some embodiments, the microcarriers comprise non-biodegradablemicrocarriers. Non-biodegradable microcapsules and microcarriersinclude, but not limited to, those made of polysulfones,poly(acrylonitrile-co-vinyl chloride), ethylene-vinyl acetate,hydroxyethylmethacrylate-methyl-methacrylate copolymers. These areuseful to provide tissue bulking properties or in embodiments where themicrocarriers are eliminated by the body.

In some embodiments, the microcarriers comprise degradable scaffolds.These include microcarriers made from naturally occurring polymers,non-limiting example of which include, among others, fibrin, casein,serum albumin, collagen, gelatin, lecithin, chitosan, alginate orpoly-amino acids such as poly-lysine. In other aspects, the degradablemicrocarriers are made of synthetic polymers, non-limiting examples ofwhich include, among others, polylactide (PLA), polyglycolide (PGA),poly(lactide-co-glycolide) (PLGA), poly(caprolactone), polydioxanonetrimethylene carbonate, polyhybroxyalkonates (e.g.,poly(hydroxybutyrate), poly(ethyl glutamate), poly(DTHiminocarbony(bisphenol A iminocarbonate), poly(ortho ester), andpolycyanoacrylates.

In some embodiments, the microcarriers comprise hydrogels, which aretypically hydrophilic polymer networks filled with water. Hydrogels havethe advantage of selective trigger of polymer swelling. Depending on thecomposition of the polymer network, swelling of the microparticle may betriggered by a variety of stimuli, including pH, ionic strength,thermal, electrical, ultrasound, and enzyme activities. Non-limitingexamples of polymers useful in hydrogel compositions include, amongothers, those formed from polymers of poly(lactide-co-glycolide);poly(N-isoproylacrylamide); poly(methacrylic acid-g-polyethyleneglycol); polyacrylic acid and poly(oxypropylene-co-oxyethylene) glycol;and natural compounds such as chrondroitan sulfate, chitosan, gelatin,fibrinogen, or mixtures of synthetic and natural polymers, for examplechitosan-poly(ethylene oxide). The polymers may be crosslinkedreversibly or irreversibly to form gels adaptable for forming threedimensional tissues.

In some embodiments, the microcarriers or beads for use in the presentinvention are composed wholly or composed partly of dextran.

In accordance with the embodiments described herein, the culturingmethod is applicable to proliferation of different types of cells,including stromal cells such as fibroblasts, and particularly primaryhuman neonatal foreskin fibroblasts. In some embodiments, the cellsinoculated onto the scaffold or framework can be stromal cellscomprising fibroblasts, with or without other cells, as furtherdescribed below. In some embodiments, the cells are stromal cells thatare typically derived from connective tissue, including, but not limitedto: (1) bone; (2) loose connective tissue, including collagen andelastin; (3) the fibrous connective tissue that forms ligaments andtendons, (4) cartilage; (5) the ECM of blood; (6) adipose tissue, whichcomprises adipocytes; and (7) fibroblasts.

Stromal cells can be derived from various tissues or organs, such asskin, heart, blood vessels, bone marrow, skeletal muscle, liver,pancreas, brain, foreskin, which can be obtained by biopsy (whereappropriate) or upon autopsy. In some embodiments, fetal fibroblasts canbe obtained in high quantity from foreskin, such as neonatal foreskins.

In some embodiments, the cells comprise fibroblasts, which can be from afetal, neonatal, adult origin, or a combination thereof. In someembodiments, the stromal cells comprise fetal fibroblasts, which cansupport the growth of a variety of different cells and/or tissues. Asused herein, a fetal fibroblast refers to fibroblasts derived from fetalsources. As used herein, neonatal fibroblast refers to fibroblastsderived from newborn sources. Under appropriate conditions, fibroblastscan give rise to other cells, such as bone cells, fat cells, and smoothmuscle cells and other cells of mesodermal origin. In some embodiments,the fibroblasts comprise dermal fibroblasts, which are fibroblastsderived from skin. Normal human dermal fibroblasts can be isolated fromneonatal foreskin. These cells are typically cryopreserved at the end ofthe primary culture.

In some embodiments, the three-dimensional tissue can be made using stemor progenitor cells, either alone, or in combination with any of thecell types discussed herein. Stem and progenitor cells include, by wayof example and not limitation, embryonic stem cells, hematopoietic stemcells, neuronal stem cells, epidermal stem cells, and mesenchymal stemcells.

In some embodiments, a “specific” three-dimensional tissue can beprepared by inoculating the three-dimensional scaffold with cellsderived from a particular organ, i.e., skin, heart, and/or from aparticular individual who is later to receive the cells and/or tissuesgrown in culture in accordance with the methods described herein.

For certain uses in vivo it is preferable to obtain the stromal cellsfrom the patient's own tissues. The growth of cells in the presence ofthe three-dimensional stromal support framework can be further enhancedby adding to the framework, or coating the framework support withproteins, e.g., collagens, laminins, elastic fibers, reticular fibers,glycoproteins; glycosaminoglycans, e.g., heparin sulfate,chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratansulfate, etc.; a cellular matrix, and/or other materials.

Thus, since the two-dimensional or three-dimensional culture systemsdescribed herein are suitable for growth of diverse cell types andtissues, and depending upon the tissue to be cultured and the collagentypes desired, the appropriate stromal cells may be selected toinoculate the framework.

While the methods and applications of the present invention are suitablefor use with different cell types, such as tissue specific cells ordifferent types of stromal cells as discussed herein, derivation of thecells for use with the present invention may also be species specific.Accordingly, ECM compositions may be generated that are speciesspecific. For example, the cells for use in the present invention mayinclude human cells. For example, the cells may be human fibroblasts.Likewise, the cells are from another species of animal, such as equine(horse), canine (dog) or feline (cat) cells. Additionally, cells fromone species or strain of species may be used to generate ECMcompositions for use in other species or related strains (e.g.,allogeneic, syngeneic and xenogeneic). It is also to be appreciated thatcells derived from various species may be combined to generatemulti-species ECM compositions.

Accordingly, the methods and compositions of the present invention aresuitable in applications involving non-human animals. As used herein,“veterinary” refers to the medical science concerned or connected withthe medical or surgical treatment of animals, especially domesticanimals. Common veterinary animals may include mammals, amphibians,avians, reptiles and fishes. For example, typical mammals may includedogs, cats, horses, rabbits, primates, rodents, and farm animals, suchas cows, horses, goats, sheep, and pigs.

As discussed above, additional cells may be present in the culture withthe stromal cells. These additional cells may have a number ofbeneficial effects, including, among others, supporting long term growthin culture, enhancing synthesis of growth factors, and promotingattachment of cells to the scaffold. Additional cell types include asnon-limiting examples, smooth muscle cells, cardiac muscle cells,endothelial cells, skeletal muscle cells, endothelial cells, pericytes,macrophages, monocytes, and adipocytes. Such cells may be inoculatedonto the framework along with fibroblasts, or in some aspects, in theabsence of fibroblasts. These stromal cells may be derived fromappropriate tissues or organs, including, by way of example and notlimitation, skin, heart, blood vessels, bone marrow, skeletal muscle,liver, pancreas, and brain. In other aspects, one or more other celltypes, excluding fibroblasts, are inoculated onto the scaffold. In stillother aspects, the scaffolds are inoculated only with fibroblast cells.

Fibroblasts may be readily isolated by disaggregating an appropriateorgan or tissue which is to serve as the source of the fibroblasts. Forexample, the tissue or organ can be disaggregated mechanically and/ortreated with digestive enzymes and/or chelating agents that weaken theconnections between neighboring cells making it possible to disperse thetissue into a suspension of individual cells without appreciable cellbreakage. Enzymatic dissociation can be accomplished by mincing thetissue and treating the minced tissue with any of a number of digestiveenzymes either alone or in combination. These include but are notlimited to trypsin, chymotrypsin, collagenase, elastase, hyaluronidase,DNase, pronase, and/or dispase etc. Mechanical disruption can also beaccomplished by a number of methods including, but not limited to theuse of grinders, blenders, sieves, homogenizers, pressure cells, orinsonators to name but a few. In some embodiments, excised foreskintissue is treated using digestive enzymes, typically collagenase and/ortrypsinase to disassociate the cells from encapsulating structures.

The isolation of fibroblasts, for example, can be carried out asfollows: fresh tissue samples are thoroughly washed and minced in Hanks'balanced salt solution (HBSS) in order to remove serum. The mincedtissue is incubated from 1-12 hours in a freshly prepared solution of adissociating enzyme such as trypsin. After such incubation, thedissociated cells are suspended, pelleted by centrifugation and platedonto culture dishes. All fibroblasts will attach before other cells,therefore, appropriate stromal cells can be selectively isolated andgrown. The isolated fibroblasts can then be grown to confluency, liftedfrom the confluent culture and inoculated onto the three-dimensionalframework, see Naughton et al., 1987, J. Med. 18(3&4):219-250.Inoculation of the three-dimensional framework with a high concentrationof stromal cells, e.g., approximately 10⁶ to 5×10⁷ cells/ml, will resultin the establishment of the three-dimensional stromal support in shorterperiods of time.

Once the tissue has been reduced to a suspension of individual cells,the suspension can be fractionated into subpopulations from which thefibroblasts and/or other stromal cells and/or elements can be obtained.This also may be accomplished using standard techniques for cellseparation including, but not limited to, cloning and selection ofspecific cell types, selective destruction of unwanted cells (negativeselection), separation based upon differential cell agglutinability inthe mixed population, freeze-thaw procedures, differential adherenceproperties of the cells in the mixed population, filtration,conventional and zonal centrifugation, centrifugal elutriation(counter-streaming centrifugation), unit gravity separation,countercurrent distribution, electrophoresis and fluorescence-activatedcell sorting. For a review of clonal selection and cell separationtechniques, see Freshney, Culture of Animal Cells. A Manual of BasicTechniques, 2d Ed., A. R. Liss, Inc., New York, 1987, Ch. 11 and 12, pp.137-168.

In some embodiments, isolated fibroblast cells can be grown to producecell banks. Cell banks are created to allow for initiating variousquantities and timing of cultivation batches and to allow preemptivetesting of cells for contaminants and specific cellular characteristics.Fibroblasts from the cell banks are subsequently grown to increase cellnumber to appropriate levels for seeding scaffolds. Operations involvingenvironmental exposure of cells and cell contacting materials areperformed by aseptic practices to reduce the potential for contaminationof foreign materials or undesirable microbes.

In some embodiments, after isolation, cells may be grown through severalpassages to a quantity suitable for building master cell banks. The cellbanks can then be, harvested and filled into appropriate vessels andpreserved in cryogenic conditions. Cells in frozen vials from mastercell banks can be thawed and grown through additional passages (usuallytwo or more). The cells can then be used to prepare cryogenicallypreserved working cell banks.

A cell expansion step uses vials of cells at the working cell bank stageto further increase cell numbers for inoculating three-dimensionalscaffolds or supports, such as mesh or microcarriers. Each passage is aseries of sub-culture steps that include inoculating cell growthsurfaces, incubation, feeding the cells and harvesting.

Cultivation for cell banks and cell expansion can be conducted byinoculating culture vessels, such as culture flasks, roller bottles ormicrocarriers. Stromal cells, such as fibroblasts, attach to theintended growth surfaces and grow in the presence of culture media.Culture vessels, such as culture flasks, roller bottles andmicrocarriers are specifically configured for cell culture and arecommonly made from various plastic materials qualified for intendedapplications. Microcarriers typically are microscopic or macroscopicbeads and are typically made of various plastic materials. However, theycan be made from other materials such as glasses or solid/semi-solidbiologically based materials such as collagens or other materials suchas Dextran, a modified sugar complex as discussed above.

During cultivation, expended media is periodically replaced with freshmedia during the course of cell growth to maintain adequate availabilityof nutrients and removal of inhibitory products of cultivation. Cultureflasks and roller bottles provide a surface for the cells to grow ontoand are typically used for cultivation of anchorage dependent cells.

In some embodiments, incubation is performed in a chamber heated at 37°C. Cultivation topologies requiring communication of media and thechamber environment use a 5% CO₂ v/v with air in the chamber gas spaceto aid in regulation of pH. Alternately, vessels equipped to maintaincultivation temperature and pH can be used for both cell expansion andECM production operations. Temperatures below 35° C. or above 38° C. andCO₂ concentrations below 3% or above 12% may not be appropriate.

Harvesting cells from attachment surfaces can conducted by removal ofgrowth media and rinsing the cells with a buffered salt solution toreduce enzyme competing protein, application of disassociating enzymesthen neutralization of the enzymes after cell detachment. Harvested cellsuspension is collected and harvest fluids are separated bycentrifugation. Cell suspensions from sub-culture harvests can besampled to assess the quantity of cells recovered and other cellularattributes and are subsequently combined with fresh media and applied asinoculums. The number of passages used for preparing cell banks andscaffold inoculum is critical with regard to achieving acceptable ECMcharacteristics.

After an appropriate three-dimensional scaffold is prepared, it isinoculated by seeding with the prepared stromal cells. Inoculation ofthe scaffold may be done in a variety of ways, such as sedimentation.Mesh prepared for culture of ECM under aerobic conditions are preparedin the same manner as for hypoxic grown mesh with the exception that ananaerobic chamber is not used to create hypoxic conditions.

For example, for both mesh prepared for culture of ECM under bothaerobic and hypoxic conditions, prepared and sterilized mesh is placedin sterile 150 mm diameter×5 mm deep petri dishes and stacked to athickness of approximately 10 pieces. Stacks of mesh are then inoculatedby sedimentation. Cells are added to fresh media to obtain theappropriate concentration of cells for inoculum. Inoculum is added tothe stack of mesh where cells settle onto the nylon fibers and attachwhile in incubated conditions. After an adequate time, individuallyseeded mesh sheets can be aseptically separated from the stack andplaced individually into separate 150 mm×15 mm petri dishes containingapproximately 50 ml of growth media.

In some embodiments, incubation of the inoculated culture is performedunder hypoxic conditions. Incubation under hypoxic conditions mayproduce an ECM and surrounding media with unique properties as comparedto ECM generated under normal culture conditions. As used herein,hypoxic conditions are characterized by a lower oxygen concentration ascompared to the oxygen concentration of ambient air (approximately15%-20% oxygen). In one aspect, hypoxic conditions are characterized byan oxygen concentration less than about 10%. In another aspect, hypoxicconditions are characterized by an oxygen concentration of about 1% to10%, 1% to 9%, 1% to 8%, 1% to 7%, 1% to 6%, 1% to 5%, 1% to 4%, 1% to3%, or 1% to 2%. In a certain aspect, the system maintains about 1-3%oxygen within the culture vessel. Hypoxic conditions can be created andmaintained by using a culture apparatus that allows one to controlambient gas concentrations, for example, an anaerobic chamber.

Incubation of cell cultures is typically performed in normal atmospherewith 15-20% oxygen and 5% CO₂ for expansion and seeding, at which pointlow oxygen cultures are split to an airtight chamber that is floodedwith 95% nitrogen/5% CO₂ so that a hypoxic environment is created withinthe culture medium.

For example, petri dishes with mesh cultured for producing ECM underhypoxic conditions are initially grown in incubation at 37° C. and 95%air/5% CO₂ for 2-3 weeks. Following the period of near atmosphericcultivation, the petri dishes of mesh are incubated in a chamberdesigned for anaerobic cultivation that is purged with a gas mixture ofapproximately 95% nitrogen and 5% CO₂. Expended growth media is replacedwith fresh media at atmospheric oxygen level through the culture periodand after media is exchanged the mesh filled petri dishes are place inthe anaerobic chamber, the chamber is purged with 95% nitrogen/5% CO₂then incubated at 37° C. Cultured mesh are harvested when they reach thedesired size or contain the desire biological components.

During the incubation period, the stromal cells will grow linearly alongand envelop the three-dimensional framework before beginning to growinto the openings of the framework. The growing cells produce a myriadof growth factors, regulatory factors and proteins, some of which aresecreted in the surrounding media, and others that are deposited on thesupport to make up the ECM more fully discussed below. Growth andregulatory factors can be added to the culture, but are not necessary.Culture of the stromal cells produces both non-soluble and solublefractions. The cells are grown to an appropriate degree to allow foradequate deposition of ECM proteins.

During culturing of the three-dimensional tissues, proliferating cellsmay be released from the framework and stick to the walls of the culturevessel where they may continue to proliferate and form a confluentmonolayer. To minimize this occurrence, which may affect the growth ofcells, released cells may be removed during feeding or by transferringthe three-dimensional cell culture to a new culture vessel. Removal ofthe confluent monolayer or transfer of the cultured tissue to freshmedia in a new vessel maintains or restores proliferative activity ofthe three-dimensional cultures. In some aspects, removal or transfersmay be done in a culture vessel which has a monolayer of cultured cellsexceeding 25% confluency. Alternatively, the culture in some embodimentsis agitated to prevent the released cells from sticking; in others,fresh media is infused continuously through the system. In some aspects,two or more cell types can be cultured together either at the same timeor one first followed by the second (e.g., fibroblasts and smooth musclecells or endothelial cells).

After inoculation of the three dimensional scaffolds, the cell cultureis incubated in an appropriate nutrient medium and incubation conditionsthat supports growth of cells into the three dimensional tissues. Manycommercially available media such as Dulbecco's Modified Eagles Medium(DMEM), RPMI 1640, Fisher's, Iscove's, and McCoy's, may be suitable forsupporting the growth of the cell cultures. The medium may besupplemented with additional salts, carbon sources, amino acids, serumand serum components, vitamins, minerals, reducing agents, bufferingagents, lipids, nucleosides, antibiotics, attachment factors, and growthfactors. Formulations for different types of culture media are describedin various reference works available to the skilled artisan (e.g.,Methods for Preparation of Media, Supplements and Substrates for SerumFree Animal Cell Cultures, Alan R. Liss, New York (1984); TissueCulture: Laboratory Procedures, John Wiley & Sons, Chichester, England(1996); Culture of Animal Cells, A Manual of Basic Techniques, 4th Ed.,Wiley-Liss (2000)).

The growth or culture media used in any of the culturing steps of thepresent invention, whether under aerobic or hypoxic conditions, mayinclude serum, or be serum free. In one aspect, the media is Dulbecco'sModified Eagle Medium with 4.5 g/L glucose, alanyl-L-glutamine, Eq 2 mM,and nominally supplemented with 10% fetal bovine serum. In anotheraspect, the media is a serum free media and is Dulbecco's Modified EagleMedium with 4.5 g/L glucose base medium with Glutamax®, supplementedwith 0.5% serum albumin, 2 μg/ml heparin, 1 μg/ml recombinant basic FGF,1 μg/ml soybean trypsin inhibitor, 1×ITS supplement(insulin-transferrin-selenium, Sigma Cat. No. 13146), 1:1000 dilutedfatty acid supplement (Sigma Cat. No. 7050), and 1:1000 dilutedcholesterol. Additionally, the same media can be used for both hypoxicand aerobic cultivation. In one aspect, the growth media is changed fromserum based media to serum free media after seeding and the first weekof growth.

Incubation conditions will be under appropriate conditions of pH,temperature, and gas (e.g., O₂, CO₂, etc) to maintain an hypoxic growthcondition. In some embodiments, the three-dimensional cell culture canbe suspended in the medium during the incubation period in order tomaximize proliferative activity and generate factors that facilitate thedesired biological activities of the fractions. In addition, the culturemay be “fed” periodically to remove the spent media, depopulate releasedcells, and add new nutrient source. During the incubation period, thecultured cells grow linearly along and envelop the filaments of thethree-dimensional scaffold before beginning to grow into the openings ofthe scaffold.

During incubation under hypoxic conditions, as compared to incubationunder normal atmospheric oxygen concentrations of about 15-20%,thousands of genes are differentially expressed. Several genes have beenfound to be upregulated or downregulated in such compositions, mostnotably certain laminin species, collagen species and Wnt factors. Invarious aspects, the three dimensional ECM may be defined by thecharacteristic fingerprint or suite of cellular products produced by thecells due to growth in hypoxic condition as compared with growth undernormal conditions. In the ECM compositions specifically exemplifiedherein, the three-dimensional tissues and surrounding media arecharacterized by expression and/or secretion of various factors.

The three dimensional tissues and compositions described herein have ECMthat is present on the scaffold or framework. In some embodiments, theECM includes various laminin and collagen types due to growth underhypoxic conditions and selection of cells grown on the support. Theproportions of ECM proteins deposited can be manipulated or enhanced byselecting fibroblasts which elaborate the appropriate collagen type aswell as growing the cells under hypoxic conditions in which expressionof specific laminin and collagen species are upregulated ordown-regulated.

Selection of fibroblasts can be accomplished in some aspects usingmonoclonal antibodies of an appropriate isotype or subclass that defineparticular collagen types. In other aspects, solid substrates such asmagnetic beads may be used to select or eliminate cells that have boundantibody. Combination of these antibodies can be used to select(positively or negatively) the fibroblasts which express the desiredcollagen type. Alternatively, the stroma used to inoculate the frameworkcan be a mixture of cells which synthesize the desired collagen types.

As discussed above, the ECM compositions described herein includevarious collagens. Accordingly, in one aspect of the present invention,the ECM composition including one or more embryonic proteins, includesupregulation of collagen species as compared with that produced inoxygen conditions of about 15-20% oxygen. In another aspect, theupregulated collagen species are type V alpha 1; IX alpha 1; IX alpha 2;VI alpha 2; VIII alpha 1; IV, alpha 5; VII alpha 1; XVIII alpha 1; andXII alpha 1.

In addition to various collagens, the ECM composition described hereininclude various laminins. Laminins are a family of glycoproteinheterotrimers composed of an alpha, beta, and gamma chain subunit joinedtogether through a coiled-coil domain. To date, 5 alpha, 4 beta, and 3gamma laminin chains have been identified that can combine to form 15different isoforms. Within this structure are identifiable domains thatpossess binding activity towards other laminin and basal laminamolecules, and membrane-bound receptors. Domains VI, IVb, and IVa formglobular structures, and domains V, IIIb, and IIIc (which containcysteine-rich EGF-like elements) form rod-like structures. Domains I andII of the three chains participate in the formation of a triple-strandedcoiled-coil structure (the long arm).

Laminin chains possess shared and unique functions and are expressedwith specific temporal (developmental) and spatial (tissue-sitespecific) patterns. The laminin alpha-chains are considered to be thefunctionally important portion of the heterotrimers, as they exhibittissue-specific distribution patterns and contain the major cellinteraction sites. Vascular endothelium is known to express two lamininisoforms, with varied expression depending on the developmental stage,vessel type, and the activation state of the endothelium.

Accordingly, in one aspect of the present invention, the ECM compositionincluding one or more embryonic proteins, includes upregulation ordownregulation of various laminin species as compared with that producedin oxygen conditions of about 15-20% oxygen.

Laminin 8 is composed of alpha-4, beta-1, and gamma-1 laminin chains.The laminin alpha-4 chain is widely distributed both in adults andduring development. In adults it can be identified in the basementmembrane surrounding cardiac, skeletal, and smooth muscle fibers, and inlung alveolar septa. It is also known to exist in the endothelialbasement membrane both in capillaries and larger vessels, and in theperineurial basement membrane of peripheral nerves, as well as inintersinusoidal spaces, large arteries, and smaller arterioles of bonemarrow. Laminin 8 is a major laminin isoform in the vascular endotheliumthat is expressed and adhered to by platelets and is synthesized in3T3-L1 adipocytes, with its level of synthesis shown to increase uponadipose conversion of the cells. Laminin 8 is thought to be the lamininisoform generally expressed in mesenchymal cell lineages to inducemicrovessels in connective tissues. Laminin 8 has also been identifiedin mouse bone marrow primary cell cultures, arteriolar walls, andintersinusoidal spaces where it is the major laminin isoform in thedeveloping bone marrow. Due to its localization in adult bone marrowadjacent to hematopoietic cells, laminin isoforms containing the alpha-4chain are likely to have biologically relevant interactions withdeveloping hematopoietic cells.

Accordingly, in another aspect of the present invention, the ECMincludes upregulation of laminin species, such as laminin 8. In anotheraspect, laminins produced by the three dimensional tissues of thepresent invention, includes at least laminin 8, which defines acharacteristic or signature of the laminin proteins present in thecomposition.

The ECM compositions described herein can include various Wnt factors.Wnt family factors are signaling molecules having roles in a myriad ofcellular pathways and cell-cell interaction processes. Wnt signaling hasbeen implicated in tumorigenesis, early mesodermal patterning of theembryo, morphogenesis of the brain and kidneys, regulation of mammarygland proliferation, and Alzheimer's disease. Accordingly, in one aspectof the present invention, the ECM composition including one or moreembryonic proteins, includes upregulation of Wnt species as comparedwith that produced in oxygen conditions of about 15-20% oxygen. Inanother aspect, the upregulated Wnt species are wnt 7a and wnt 11. Inanother aspect, Wnt factors produced by the three dimensional tissues ofthe present invention, include at least wnt7a, and wntl 1, which definesa characteristic or signature of the Wnt proteins present in thecomposition.

The culturing methods described herein, including culture under hypoxicconditions, have also been shown to upregulate expression of variousgrowth factors. Accordingly, the ECM compositions described herein caninclude various growth factors, such as a vascular endothelial growthfactor (VEGF). As used herein, a VEGF in intended to include all knownVEGF family members. VEGFs are a sub-family of growth factors, morespecifically of platelet-derived growth factor family of cystine-knotgrowth factors. VEGFs have a well known role in both vasculogenesis andangiogenesis. Several VEGFs are known, including VEGF-A, which wasformerly known as VEGF before the discovery of other VEGF species. OtherVEGF species include placenta growth factor (PlGF), VEGF-B, VEGF-C andVEGF-D. Additionally, several isoforms of human VEGF are well known.

In accordance with the increased production of Wnt proteins as well asgrowth factors by culturing under hypoxic conditions as describedherein, the present invention further provides a method of producing aWnt protein and a vascular endothelial growth factor (VEGF). The methodcan include culturing cells under hypoxic conditions as describedherein, on a three-dimensional surface in a suitable growth medium, toproduce the Wnt protein and the VEGF. In an exemplary aspect, the Wntspecies are wnt 7a and wnt 11 and the VEGF is VEGF-A. The proteins maybe further processed or harvested as described further herein or bymethods known in the art.

The ECM compositions of the present invention may include both solubleand non-soluble fractions or any portion thereof. It is to be understoodthat the compositions of the present invention may include either orboth fractions, as well as any combination thereof. Additionally,individual components may be isolated from the fractions to be usedindividually or in combination with other isolates or knowncompositions.

Accordingly, in various aspects, ECM compositions produced using themethods of the present invention may be used directly or processed invarious ways, the methods of which may be applicable to both thenon-soluble and soluble fractions. The soluble fraction, including thecell-free supernatant and media, may be subject to lyophilization forpreserving and/or concentrating the factors. Various biocompatiblepreservatives, cryoprotectives, and stabilizer agents may be used topreserve activity where required. Examples of biocompatible agentsinclude, among others, glycerol, dimethyl sulfoxide, and trehalose. Thelyophilizate may also have one or more excipients such as buffers,bulking agents, and tonicity modifiers. The freeze-dried media may bereconstituted by addition of a suitable solution or pharmaceuticaldiluent, as further described below.

In other aspects, the soluble fraction is dialyzed. Dialysis is one ofthe most commonly used techniques to separate sample components based onselective diffusion across a porous membrane. The pore size determinesmolecular-weight cutoff (MWCO) of the membrane that is characterized bythe molecular-weight at which 90% of the solute is retained by themembrane. In certain aspects membranes with any pore size iscontemplated depending on the desired cutoff. Typical cutoffs are 5,000Daltons, 10,000 Daltons, 30,000 Daltons, and 100,000 Daltons, howeverall sizes are contemplated.

In some aspects, the soluble fraction may be processed by precipitatingthe active components (e.g., growth factors) in the media. Precipitationmay use various procedures, such as salting out with ammonium sulfate oruse of hydrophilic polymers, for example polyethylene glycol.

In other aspects, the soluble fraction is subject to filtration usingvarious selective filters. Processing the soluble fraction by filteringis useful in concentrating the factors present in the fraction and alsoremoving small molecules and solutes used in the soluble fraction.Filters with selectivity for specified molecular weights include <5000Daltons, <10,000 Daltons, and <15,000 Daltons. Other filters may be usedand the processed media assayed for therapeutic activity as describedherein. Exemplary filters and concentrator system include those basedon, among others, hollow fiber filters, filter disks, and filter probes(see, e.g., Amicon Stirred Ultrafiltration Cells).

In still other aspects, the soluble fraction is subject tochromatography to remove salts, impurities, or fractionate variouscomponents of the medium. Various chromatographic techniques may beemployed, such as molecular sieving, ion exchange, reverse phase, andaffinity chromatographic techniques. For processing conditioned mediumwithout significant loss of bioactivity, mild chromatographic media maybe used. Non-limiting examples include, among others, dextran, agarose,polyacrylamide based separation media (e.g., available under varioustradenames, such as Sephadex, Sepharose, and Sephacryl).

In still other aspects, the conditioned media is formulated asliposomes. The growth factors may be introduced or encapsulated into thelumen of liposomes for delivery and for extending life time of theactive factors. As known in the art, liposomes can be categorized intovarious types: multilamellar (MLV), stable plurilamellar (SPLV), smallunilamellar (SUV) or large unilamellar (LUV) vesicles. Liposomes can beprepared from various lipid compounds, which may be synthetic ornaturally occurring, including phosphatidyl ethers and esters, such asphosphotidylserine, phosphotidylcholine, phosphatidyl ethanolamine,phosphatidylinositol, dimyristoylphosphatidylcholine; steroids such ascholesterol; cerebrosides; sphingomyelin; glycerolipids; and otherlipids (see, e.g., U.S. Pat. No. 5,833,948).

The soluble fraction may be used directly without additional additives,or prepared as pharmaceutical compositions with various pharmaceuticallyacceptable excipients, vehicles or carriers. A “pharmaceuticalcomposition” refers to a form of the soluble and/or non-solublefractions and at least one pharmaceutically acceptable vehicle, carrier,or excipient. For intradermal, subcutaneous or intramuscularadministration, the compositions may be prepared in sterile suspension,solutions or emulsions of the ECM compositions in aqueous or oilyvehicles. The compositions may also contain formulating agents, such assuspending, stabilizing or dispersing agents. Formulations for injectionmay be presented in unit dosage form, ampules in multidose containers,with or without preservatives. Alternatively, the compositions may bepresented in powder form for reconstitution with a suitable vehicleincluding, by way of example and not limitation, sterile pyrogen freewater, saline, buffer, or dextrose solution.

In other aspects, the three dimensional tissues are cryopreservedpreparations, which are thawed prior to use. Pharmaceutically acceptablecryopreservatives include, among others, glycerol, saccharides, polyols,methylcellulose, and dimethyl sulfoxide. Saccharide agents includemonosaccharides, disaccharides, and other oligosaccharides with glasstransition temperature of the maximally freeze-concentrated solution(Tg) that is at least −60, −50, −40, −30, −20, −10, or 0° C. Anexemplary saccharide for use in cryopreservation is trehalose.

In some aspects, the three dimensional tissues are treated to kill thecells prior to use. In some aspects, the ECM deposited on the scaffoldsmay be collected and processed for administration (see U.S. Pat. Nos.5,830,708 and 6,280,284, incorporated herein by reference).

In some embodiments, the three dimensional tissue may be concentratedand washed with a pharmaceutically acceptable medium for administration.Various techniques for concentrating the compositions are available inthe art, such as centrifugation or filtering. Examples include, dextransedimentation and differential centrifugation. Formulation of the threedimensional tissues may also involve adjusting the ionic strength of thesuspension to isotonicity (i.e., about 0.1 to 0.2) and to physiologicalpH (i.e., pH 6.8 to 7.5). The formulation may also contain lubricants orother excipients to aid in administration or stability of the cellsuspension. These include, among others, saccharides (e.g., maltose) andorganic polymers, such as polyethylene glycol and hyaluronic acid.Additional details for preparation of various formulations are describedin U.S. Patent Publication No. 2002/0038152, incorporated herein byreference.

As discussed above, the ECM compositions of the present invention may beprocessed in a number of ways depending on the anticipated applicationand appropriate delivery or administration of the ECM composition. Theterms “administration” or “administering” are defined to include an actof providing a compound or pharmaceutical composition of the inventionto a subject in need of treatment. In some embodiments, the compositionis administered topically on the skin.

The term “subject” as used herein refers to any individual or patient towhich the subject methods are performed. Generally the subject is human,although as will be appreciated by those in the art, the subject may bean animal. Thus other animals, including mammals such as rodents(including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits,farm animals including cows, horses, goats, sheep, pigs, etc., andprimates (including monkeys, chimpanzees, orangutans and gorillas) areincluded within the definition of subject.

The ECM compositions of the present invention have a variety ofapplications including, but not limited to, promoting repair and/orregeneration of damaged cells or tissues, use in patches to promotetissue regeneration, use in tissue culture systems for culturing cells,such as stem cells, use in surface coatings used in association withimplantable devices (e.g., pacemakers, stents, stent grafts, vascularprostheses, heart valves, shunts, drug delivery ports or catheters),promoting soft tissue repair, augmentation, and/or improvement of a skinsurface, such as wrinkles, use as a biological anti-adhesion agent or asa biological vehicle for cell delivery or maintenance at a site ofdelivery.

Additionally, the ECM compositions derived from culturing cells asdescribed in any method herein, may be used in any other application ormethod of the present invention. For example, the ECM compositionsgenerated by culturing cells using the tissue culture system of thepresent invention may be used, for example, in the repair and/orregeneration of cells, use in patches to promote tissue regeneration,use in tissue culture systems for culturing cells, such as stem cells,use in surface coatings used in association with implantable devices(e.g., pacemakers, stents, stent grafts, vascular prostheses, heartvalves, shunts, drug delivery ports or catheters), promoting soft tissuerepair, augmentation, and/or improvement of a skin surface, such aswrinkles, use as a biological anti-adhesion agent or as a biologicalvehicle for cell delivery or maintenance at a site of delivery.

In various embodiments of any one of each or any of the above- orbelow-mentioned embodiments, methods for repair and/or regeneration ofcells or tissue and promoting soft tissue repair are provided. Oneembodiment includes a method of repair and/or regeneration of cells bycontacting cells to be repaired or regenerated with the ECM compositionsof the present invention. The method may be used for repair and/orregeneration of a variety of cells as discussed herein, includingosteochondral cells.

In some embodiments, formulations made with the ECM compositions may beused to treat aging skin of an individual. The skin of the individualmay have loss of proteostasis, skin wounds, hyperpigmentation, loose orsagging skin, deep wrinkles, and/or fine lines. In some embodiments, theformulation for treating aging skin comprises ECM compositions made bythe methods described herein. In some embodiments, the formulation fortreating aging skin further comprises botanicals. In some embodiments,the formulation for treating aging skin further comprises marineextracts. In some embodiments, the formulation for treating aging skinfurther comprises peptides.

In one aspect, the method contemplates repair of osteochondral defects.As used herein, “osteochondral cells” refers to cells which belong toeither the chondrogenic or osteogenic lineage or which can undergodifferentiation into either the chondrogenic or osteogenic lineage,depending on the environmental signals. This potential can be tested invitro or in vivo by known techniques. Thus, in one aspect, the ECMcompositions of the present invention are used to repair and/orregenerate, chondrogenic cells, for example, cells which are capable ofproducing cartilage or cells which themselves differentiate into cellsproducing cartilage, including chondrocytes and cells which themselvesdifferentiate into chondrocytes (e.g., chondrocyte precursor cells).Thus, in another aspect, the compositions of the present invention areuseful in repair and/or regeneration of connective tissue. As usedherein, “connective tissue” refers to any of a number of structuraltissues in the body of a mammal including but not limited to bone,cartilage, ligament, tendon, meniscus, dermis, hyperdermis, muscle,fatty tissue, joint capsule.

The ECM compositions of the present invention may be used for treatingdefects or injury on the skin. The composition may be used for improvingthe quality of the skin on the individual. In some embodiments, thecomposition is used on the skin to improve the aesthetic appearance ofthe skin. In some embodiments, the composition leads to healing of theskin.

As discussed above, growth factors or other biological agents whichinduce or stimulate growth of particular cells may be included in theECM compositions of the present invention. The type of growth factorswill be dependent on the cell-type and application for which thecomposition is intended. For example, in the case of osteochondralcells, additional bioactive agents may be present such as cellulargrowth factors (e.g., TGF-β), substances that stimulate chondrogenesis(e.g., BMPs that stimulate cartilage formation such as BMP-2, BMP-12 andBMP-13), factors that stimulate migration of stromal cells to thescaffold, factors that stimulate matrix deposition, anti-inflammatories(e.g., non-steroidal anti-inflammatories), immunosuppressants (e.g.,cyclosporin). Other proteins may also be included, such as other growthfactors such as platelet derived growth factors (PDGF), insulin-likegrowth factors (IGF), fibroblast growth factors (FGF), epidermal growthfactor (EGF), human endothelial cell growth factor (ECGF), granulocytemacrophage colony stimulating factor (GM-CSF), vascular endothelialgrowth factor (VEGF), cartilage derived morphogenetic protein (CDMP),other bone morphogenetic proteins such as OP-1, OP-2, BMP3, BMP4, BMP9,BMP11, BMP14, DPP, Vg-1, 60A, and Vgr-1, collagens, elastic fibers,reticular fibers, glycoproteins or glycosaminoglycans, such as heparinsulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate,keratin sulfate, etc. For example, growth factors such as TGF-13, withascorbate, have been found to trigger chondrocyte differentiation andcartilage formation by chondrocytes. In addition, hyaluronic acid is agood substrate for the attachment of chondrocytes and other stromalcells and can be incorporated as part of the scaffold or coated onto thescaffold.

Additionally, other factors which influence the growth and/or activityof particular cells may also be used. For example, in the case ofchondrocytes, a factor such as a chondroitinase which stimulatescartilage production by chondrocytes can be added to the matrix in orderto maintain chondrocytes in a hypertrophic state as described in U.S.Patent Application No. 2002/0122790, incorporated herein by reference.In some embodiments, the methods include the presence of polysulphatedalginates or other polysulphated polysaccharides such as polysulphatedcyclodextrin and/or polysulphated inulin, or other components capable ofstimulating production of ECM of connective tissue cells as described inInternational Patent Publication No. WO 2005/054446, incorporated hereinby reference.

The cell or tissue to be repaired and/or regenerated may be contacted invivo or in vitro by any of the methods described herein. For example,the ECM compositions may be applied topically onto the skin of thesubject. In another aspect, the tissue or cells to be repaired and/orregenerated may be harvested from the subject and cultured in vitro andsubsequently implanted or administered to the subject using knownsurgical techniques.

As discussed above, the ECM compositions of the present invention may beprocessed in a variety of ways. Accordingly, in one embodiment, thepresent invention includes a tissue culture system. In various aspects,the culture system is composed of the ECM compositions described herein.The ECM compositions of the present invention may be incorporated intothe tissue culture system in a variety of ways. For example,compositions may be incorporated as coatings, by impregnatingthree-dimensional scaffold materials as described herein, or asadditives to media for culturing cells. Accordingly, in one aspect, theculture system can include three-dimensional support materialsimpregnated with any of the ECM compositions described herein, such asgrowth factors or embryonic proteins.

The ECM compositions described herein may serve as a support orthree-dimensional support for the growth of various cell types. Any celltype capable of cell culture is contemplated. In one aspect, the culturesystem can be used to support the growth of stem cells. In anotheraspect, the stem cells are embryonic stem cells, mesenchymal stem cellsor neuronal stem cells.

The tissue culture system may be used for generating additional ECMcompositions, such as implantable tissue. Accordingly, culturing ofcells using the tissue culture system of the present invention may beperformed in vivo or in vitro. For example, the tissue culture system ofthe present invention may be used to generate ECM compositions forinjection or implantation into a subject. The ECM compositions generatedby the tissue culture system may be processed and used in any methoddescribed herein.

The ECM compositions of the present invention may be used as abiological vehicle for cell delivery. As described herein, the ECMcompositions may include both soluble and/or non-soluble fractions. Assuch, in another embodiment of the present invention, a biologicalvehicle for cell delivery or maintenance at a site of delivery includingthe ECM compositions of the present invention, is described. The ECMcompositions of the present invention, including cells andthree-dimensional tissue compositions, may be used to promote and/orsupport growth of cells in vivo. The vehicle can be used in anyappropriate application, for example to support injections of cells,such as stem cells, into damaged heart muscle or for tendon and ligamentrepair as described above.

Appropriate cell compositions (e.g., isolated ECM cells of the presentinvention and/or additional biological agents) can be administeredbefore, after or during the ECM compositions are administered. Forexample, the cells can be seeded into the site of administration,defect, and/or implantation before the culture system or biologicaldelivery vehicle is implanted into the subject. Alternatively, theappropriate cell compositions can be administered after (e.g., byinjection into the site). The cells act therein to induce tissueregeneration and/or cell repair.

ECM compositions have been described for promoting angiogenesis inorgans and tissues by administering such compositions to promoteendothelialization and vascularization in the heart and related tissues.Accordingly, in yet another embodiment, the present invention applyingthe ECM composition topically. The preparation and use of ECMcompositions grown under normal oxygen conditions is described in U.S.Patent Application No. 2004/0219134 incorporated herein by reference.

In some embodiments, various implantable devices and tissue regenerationpatches including the ECM compositions described herein which allow forbenefits, such as tissue ingrowth are provided. As discussed herein, theECM compositions may serve as coatings on medical devices, such aspatches or other implantable devices. In some embodiments, such devicesare useful for wound repair.

A variety of techniques are known in the art for applying biologicalcoatings to medical device surfaces that may be utilized with thepresent invention. For example, ECM compositions may be coated usingphotoactive crosslinkers allowing for permanent covalent bonding todevice surfaces upon activation of the crosslinker by applyingultraviolet radiation. An exemplary crosslinker is TriLite™ crosslinker,which has been shown to be non-cytotoxic, non-irritating to biologicaltissue and non-sensitizing. ECM materials may be unseparated orseparated into individual components, such as human collagens,hyaluronic acid (HA), fibronectin, and the like before coating orincorporation into various implantable devices. Further, additionalgrowth factors and such may be incorporated to allow for beneficialimplantation characteristics, such as improved cell infiltration.

In various related embodiments, methods and devices applicable incosmetic and cosmeceutical applications, such as, but not limited toanti-aging, anti-wrinkle, skin fillers, moisturizers, pigmentationaugmentation, skin firming, and the like, are provided. Accordingly, inone embodiment, the present invention includes a method for improvementof a skin surface in a subject including administering to the subject atthe site of a wrinkle, the ECM compositions described herein. In arelated embodiment, the present invention includes a method for softtissue repair or augmentation in a subject including administering tothe subject at the site of a wrinkle, the ECM compositions describedherein. In various cosmetic applications, the compositions may beformulated as appropriate, such as topical formulations. In someembodiments, ECM compositions formulated as topicals have been shown tobe effective in various skin aesthetics applications, such asanti-wrinkle, anti-aging applications as well as an adjunct to ablativelaser surgery. Several beneficial characteristics of ECM containingtopicals have been shown. Such benefits include 1) facilitatingre-epithelization following resurfacing; 2) reduction of non-ablativeand ablative fractional laser resurfacing symptoms (e.g., erythema,edema, crusting, and sensorial discomfort); 3) generating smooth, eventextured skin; 4) generating skin moisturization; 5) reducing appearanceof fine lines/wrinkles; 6) increasing skin firmness and suppleness; 7)reducing skin dyspigmentation; and 8) reducing red, blotchy skin.

The compositions of the present invention may be prepared as known inthe art, however employing the innovative culture methods describedherein (e.g., culture under hypoxic conditions). The preparation and useof ECM compositions created under normal oxygen culture conditions forthe repair and/or regeneration of cells, improvement of skin surfaces,and soft tissue repair are described in U.S. Pat. No. 5,830,708, U.S.Pat. No. 6,284,284, U.S. Patent Application No. 2002/0019339 and U.S.Patent Application No. 2002/0038152, all incorporated herein byreference.

The compositions or active components used herein, will generally beused in an amount effective to treat or prevent the particular defect,skin condition and/or disease being treated. The compositions may beadministered therapeutically to achieve therapeutic benefit orprophylactically to achieve prophylactic benefit. Therapeutic benefitincludes the eradication or amelioration of an underlying defect, skincondition, disorder and/or disease being treated. Therapeutic benefitalso includes halting or slowing the progression of the defect, skincondition, disorder and/or disease, regardless of whether improvement isrealized.

The amount of the composition administered will depend upon a variety offactors, including, for example, the type of composition, the particularindication being treated, the mode of administration, whether thedesired benefit is prophylactic or therapeutic, the severity of theindication being treated and the age and weight of the patient, andeffectiveness of the dosage form. Determination of an effective dosageis well within the capabilities of those skilled in the art.

Initial dosages may be estimated initially from in vitro assays. Initialdosages can also be estimated from in vivo data, such as animal models.Animals models useful for testing the efficacy of compositions forenhancing hair growth include, among others, rodents, primates, andother mammals. The skilled artisans can determine dosages suitable forhuman administration by extrapolation from the in vitro and animal data.

Dosage amounts will depend upon, among other factors, the activity ofthe conditioned media, the mode of administration, the condition beingtreated, and various factors discussed above. Dosage amount and intervalmay be adjusted individually to provide levels sufficient to themaintain the therapeutic or prophylactic effect.

Presented below are examples discussing generation of ECM compositionscontemplated for the discussed applications. The following examples areprovided to further illustrate the embodiments of the present invention,but are not intended to limit the scope of the invention. While they aretypical of those that might be used, other procedures, methodologies, ortechniques known to those skilled in the art may alternatively be used.

Methods of Making a Composition

In some aspects, a method of producing a composition for improvement ofa tissue in a subject is provided. The method comprises culturingfibroblast cells under hypoxic conditions on microcarrier beads or athree dimensional surface in a suitable growth medium, under 1-5%oxygen, thereby producing multipotent stem cells, wherein themultipotent stem cells produce and secrete into a growth medium acomposition that promotes repair and regeneration of damaged tissue whenadministered to the region of tissue in need of repair in the subjectand collecting the growth medium, thereby producing the composition. Insome embodiments, additives to the composition comprise at least onebotanical, at least one extract, at least one peptide, at least oneprotein and/or at least one marine extract. In some embodiments, theculturing is performed on microcarrier beads or a three dimensionalsurface in a suitable growth medium. In some embodiments, the culturingis performed for at least two weeks. In some embodiments, the growthmedium is collected after two weeks. In some embodiments, the methodfurther comprises adding a seed extract to the composition. In someembodiments, the method further comprises adding a marine extract to thecomposition. In some embodiments, the method further comprises adding abacterial ferment to the composition. In some embodiments, the growthmedium further comprises stem cell factors. In some embodiments, thegrowth medium further comprises cytokines. In some embodiments, thegrowth medium further comprises at least one matrix protein. In someembodiments, the at least one peptide comprises dimer tripeptide 43and/or trifluoroacetyl tripeptide-2. In some embodiments, the subject isin need of tissue repair. In some embodiments, the subject has fine andor deep wrinkles. In some embodiments, the subject exhibits tactileand/or skin roughness of the tissue. In some embodiments, the subjecthas hyperpigmentation. In some embodiments, the subject has photodamage.In some embodiments, the subject lacks evenness in pigmentation and/orskin tone. In some embodiments, the subject has a skin coloring on theFitzpatrick scale of 1, 2, 3, 4 or 5.

Compositions

A composition made by the method of any one of the embodiments of anyone of each or any of the above- or below-mentioned embodiments for usein treating a subject is provided.

In some embodiments, the composition further comprises at least onebotanical.

In some embodiments, the composition further comprises at least oneextract.

In some embodiments, the composition further comprises at least onepeptide.

In some embodiments, the composition further comprises a seed extract.

In some embodiments, the composition further comprises a marine extract.

In some embodiments, the composition further comprises a bacterialferment.

In some embodiments, the composition further comprises stem cellfactors.

In some embodiments, the composition further comprises cytokines.

In some embodiments, the composition further comprises at least onematrix protein.

In some embodiments, the at least one peptide comprises dimer tripeptide43 and/or trifluoroacetyl tripeptide-2.

In some embodiments, a composition is provided comprising: growthmedium, wherein the growth medium is collected from multipotent stemcells produced under hypoxic conditions; at least one botanical, atleast one extract, and at least one peptide.

In some embodiments, the composition is odorless.

In some embodiments, the composition is clear.

Methods of Treatment

In some embodiments, a method of improving the appearance of anindividual is provided, the method comprising topically applying thecomposition of any one of embodiments herein, onto a tissue of a subjectto thereby improve the aesthetic quality of the tissue.

In some embodiments, the subject has fine or deep wrinkles.

In some embodiments, the subject has tactile and/or skin roughness ofthe tissue.

In some embodiments, the subject has loose or sagging skin.

In some embodiments, the method improves roughness of skin.

In some embodiments, the method restores volume moisture to the tissue.

In some embodiments, the method results in reduced inflammatory responseof the tissue.

In some embodiments, the subject is in need of tissue repair.

In some embodiments, the subject is suffering from a burn wound.

In some embodiments, the method leads to healing of the tissue.

In some embodiments, skin is protected from free radical damage.

In some embodiments, the composition supports epidermal cell-celladhesion.

In some embodiments, the composition supports a dermal epidermaljunction.

In some embodiments, the composition supports stem cells function andproliferation.

In some embodiments, the composition supports intercellularcommunication.

In some embodiments, the composition supports cellular recycling andprotein homeostasis.

In some embodiments, the composition prevents cellular senescence.

In some embodiments, the composition supports collagen, elastin and ECMcomponents.

In some embodiments, the composition supports heparan sulfate andproteoglycans.

In some embodiments, the composition increases expression of stemnessbiomarkers.

In some embodiments, the composition provides comprehensive skinrejuvenation at the epidermis, dermal-epidermal junction and the dermis.

In some embodiments, application of the composition increases geneexpression of proliferation and myofibroblast markers.

In some embodiments, application of the composition increases expressionof stemness biomarkers.

In some embodiments, application of the composition improves theappearance of fine lines and deep wrinkles.

Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the invention so claimed areinherently or expressly described and enabled herein.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above-citedreferences and printed publications are individually incorporated hereinby reference in their entirety.

It is to be understood that the embodiments of the invention disclosedherein are illustrative of the principles of the present invention.Other modifications that may be employed are within the scope of theinvention. Thus, by way of example, but not of limitation, alternativeconfigurations of the present invention may be utilized in accordancewith the teachings herein. Accordingly, the present invention is notlimited to that precisely as shown and described.

What is claimed is:
 1. A composition for repair and regeneration of skintissue on a subject, the composition comprising; a conditioned mediumcollected from culturing human fibroblast cells under hypoxic conditionsin a suitable cell culture medium; and at least one additive; whereinthe conditioned medium is stored in a first container and the additiveis stored in a second container, and wherein the conditioned medium andthe additive are mixed prior to application to the skin tissue.
 2. Thecomposition of claim 1, wherein the first container and the secondcontainer are separate chambers in a dual chamber container.
 3. Thecomposition of claim 2, wherein the human fibroblast cells are culturedunder hypoxic conditions on a substrate grown in a suitable cell culturemedium under 1-5% oxygen thereby producing embryo-like properties. 4.The composition of claim 3, wherein the substrate is microcarrier beadsor a three-dimensional surface.
 5. The composition of claim 2, whereinthe composition further comprises at least one botanical or botanicalextract.
 6. The composition of claim 5, wherein the at least onebotanical or botanical extract is present within a range of about 0.5 toabout 2.0% by weight of the composition.
 7. The composition of claim 2,wherein the composition further comprises at least one peptide.
 8. Thecomposition of claim 7, wherein the at least one peptide is presentwithin a range of about 0.0001 to about 0.001% by weight of thecomposition.
 9. The composition of claim 2, wherein the compositionfurther comprises at least one seed extract.
 10. The composition ofclaim 9, wherein the at least one seed extract is present within a rangeof about 0.5 to about 2% by weight of the composition.
 11. Thecomposition of claim 2, wherein the composition further comprises atleast one marine extract.
 12. The composition of claim 11, wherein theat least one marine extract is present within a range of about 0.01 toabout 0.1% by weight of the composition.
 13. The composition of claim 2,wherein the composition further comprises at least one bacterialferment.
 14. The composition of claim 13, wherein the at least onebacterial ferment is present within a range of about 0.5 to about 3.0%by weight of the composition.
 15. The composition of claim 2, whereinthe composition further comprises stem cell factors.
 16. The compositionof claim 15, wherein the stem cell factors are present within a range ofabout 0.1 to about 30% by weight of the composition.
 17. The compositionof claim 2, wherein the composition further comprises at least onecytokine.
 18. The composition of claim 2, wherein the composition iscolorless.
 19. A method of producing a composition for improvement of atissue in a subject, the method comprising: culturing human fibroblastcells under hypoxic conditions on microcarrier beads or a threedimensional surface in a suitable cell culture medium, under 1-5%oxygen, thereby producing cells having embryo-like properties, whereinthe cells having embryo-like properties produce and secrete into agrowth medium a composition that promotes repair and regeneration ofdamaged tissue when administered to the region of tissue in need ofrepair in the subject; and collecting the cell culture medium; andadding an additive to the cell culture medium to produce thecomposition.
 20. The method of claim 19, further comprising adding atleast one extract to the composition.
 21. The method of claim 19,further comprising adding at least one botanical or botanical extract tothe composition.
 22. The method of claim 19, further comprising addingat least one peptide to the composition.
 23. The method of claim 19,wherein the culturing is performed for at least two weeks.
 24. Themethod of claim 19, further comprising adding a seed extract to thecomposition.
 25. The method of claim 19, further comprising adding amarine extract to the composition.
 26. The method of claim 19, furthercomprising adding a bacterial ferment to the composition.
 27. The methodof claim 19, further comprising cytokines.
 28. The method of claim 19,further comprising adding at least one extracellular matrix protein. 29.The method of claim 22, wherein the at least one peptide comprises dimertripeptide 43 and/or trifluoroacetyl tripeptide-2.
 30. The method ofclaim 19, wherein the subject has fine or deep wrinkles.
 31. The methodof claim 19, wherein the subject exhibits tactile or skin roughness ofthe issue.
 32. The method of claim 19, wherein the subject hashyperpigmentation.
 33. The method of claim 19, wherein the subject hasphotodamage.
 34. The method of claim 19, wherein the subject lacksevenness in pigmentation or skin tone.
 35. The method of claim 19,wherein the subject has a skin coloring on the Fitzpatrick scale of 1,2, 3, 4 or
 5. 36. The method of claim 19, wherein the composition isstored in a one chamber container.
 37. A composition made by the methodof claim 19 for use in treating a subject.
 38. A composition comprising:a growth medium, wherein the growth medium is collected from multipotentstem cells produced under hypoxic conditions; at least one botanical; atleast one extract; and at least one peptide.
 39. The composition ofclaim 38, wherein the composition is odorless.
 40. The composition ofclaim 38, wherein the composition is clear.
 41. A method of improvingthe appearance of a subject, the method comprising topically applyingthe composition of claim 38 onto a tissue of a subject to therebyimprove the aesthetic quality of the tissue.
 42. The method of claim 41,wherein the subject has fine or deep wrinkles.
 43. The method of claim42, wherein the subject has tactile roughness of the tissue.
 44. Themethod of claim 41, wherein the subject has loose or sagging skin. 45.The method of claim 41, wherein the method improves roughness of skin.46. The method of claim 41, wherein the method restores volume moistureto the tissue.
 47. The method of claim 41, wherein the method results inreduced inflammatory response of the tissue.
 48. The method of claim 41,wherein the subject is suffering from a burn wound.
 49. The method ofclaim 41, wherein the skin is protected from free radical damage. 50.The method of claim 41, wherein the composition supports epidermalcell-cell adhesion.
 51. The method of claim 41, wherein the compositionsupports a dermal epidermal junction.
 52. The method of claim 41,wherein the composition supports stem cells function and proliferation.53. The method of claim 41, wherein the composition supportsintercellular communication.
 54. The method of claim 41, wherein thecomposition supports cellular recycling and protein homeostasis.
 55. Themethod of claim 41, wherein the composition prevents cellularsenescence.
 56. The method of claim 41, wherein the composition supportscollagen, elastin and other ECM components.
 57. The method of claim 41,wherein the composition supports heparan sulfate and proteoglycans.